Funatsu T, Anazawa T, Ishiwata S
Yanagida Biomotron Project, ERATO, JRDC, Osaka, Japan.
J Muscle Res Cell Motil. 1994 Apr;15(2):158-71. doi: 10.1007/BF00130426.
Thin filaments were reconstituted by incorporating exogenous actin, tropomyosin and troponin into glycerinated skeletal muscle fibres or myofibrils. Firstly, thin filaments except short fragments at the Z line were selectively removed by treatment with plasma gelsolin, an actin severing protein. As a result, the fibres (or fibrils) lost the ability to generate active tension. Next, actin filaments were reconstituted by adding purified G-actin which polymerizes onto the actin fragments which remained at the Z line. Rhodamine phalloidin staining of myofibrils showed that exogenous actin was incorporated into the position where the intrinsic thin filaments located. Thin section electron micrographs of fibres showed that reconstituted actin filaments ran from the Z line to the inside of the A band, with some reaching the H zone. The number density of reconstituted actin filaments in the A band was about 20% of that found in intact fibres. The actin filament-reconstituted fibres (or fibrils) generated active tension in a Ca(2+)-insensitive manner and the tension was reversibly suppressed by 2,3-butanedione 2-monoxime. The recovered active tension was about 20% of tension developed by intact fibres. These results indicate that reconstituted actin filaments bear active tension similar to that borne by intact thin filaments. Thin filament-reconstituted fibres, which were prepared by adding purified tropomyosin-troponin complexes into actin filament-reconstituted fibres, showed Ca(2+)-sensitive tension generation. The maximum tension generated was not affected by the presence of tropomyosin and troponin. SDS-PAGE analysis showed that more than 25% of actin and 20% of tropomyosin and troponin was incorporated into the reconstituted fibres. These results indicate that the structure and function of thin filaments are substantially reconstituted by self-assembly of actin, tropomyosin and troponin. The reconstituted fibres and fibrils will be useful for studying the molecular mechanism of muscle contraction and its regulation.
通过将外源性肌动蛋白、原肌球蛋白和肌钙蛋白整合到甘油化骨骼肌纤维或肌原纤维中，重新构建了细肌丝。首先，用血浆凝溶胶蛋白（一种肌动蛋白切割蛋白）处理，选择性地去除了Z线处短片段以外的细肌丝。结果，纤维（或肌原纤维）失去了产生主动张力的能力。接下来，通过添加纯化的G-肌动蛋白来重新构建肌动蛋白丝，G-肌动蛋白会聚合到保留在Z线处的肌动蛋白片段上。肌原纤维的罗丹明鬼笔环肽染色表明，外源性肌动蛋白被整合到固有细肌丝所在的位置。纤维的超薄切片电子显微镜图像显示，重新构建的肌动蛋白丝从Z线延伸到A带内部，有些甚至到达H区。A带中重新构建的肌动蛋白丝的数量密度约为完整纤维中的20%。肌动蛋白丝重构的纤维（或肌原纤维）以Ca(2+)不敏感的方式产生主动张力，并且张力被2,3-丁二酮单肟可逆抑制。恢复的主动张力约为完整纤维产生张力的20%。这些结果表明，重新构建的肌动蛋白丝承受的主动张力与完整细肌丝承受的相似。通过向肌动蛋白丝重构的纤维中添加纯化的原肌球蛋白-肌钙蛋白复合物制备的细肌丝重构纤维，表现出Ca(2+)敏感的张力产生。产生的最大张力不受原肌球蛋白和肌钙蛋白存在的影响。SDS-PAGE分析表明，超过25%的肌动蛋白以及20%的原肌球蛋白和肌钙蛋白被整合到重构纤维中。这些结果表明，细肌丝的结构和功能通过肌动蛋白、原肌球蛋白和肌钙蛋白的自组装得到了实质性的重构。重构的纤维和肌原纤维将有助于研究肌肉收缩及其调节的分子机制。
